Characterization of the ocular inflammatory response to AAV reveals divergence by sex and age

Recombinant adeno-associated virus (AAV) is the leading platform for gene therapy in ocular disease. Progress for gene therapy has been hindered by AAV-induced inflammation, which limits dose escalation and long-term efficacy. Characterisation of the ocular immune response to AAV in mice has been restricted to young animals and demonstrate activation of microglia, antigen presentation and a dominant T cell lymphocyte infiltration. The extent of the inflammatory response alters with age and sex and these factors have not been fully represented in the pre-clinical development of ocular AAV gene therapies. Here, we intravitreally inject a null AAV2 vector in young (3-month), middle aged (9-month) and old (18-month) Cx3cr1-creER:R26tdTomato+/- mice of both sexes. Applying clinical imaging, flow cytometric analyses and bulk-sequencing of sorted resident microglia we interrogate the impact of sex and age on the longitudinal response of both microglia and infiltrating cellular response to AAV. Young animals have a dynamic response, with a peak in inflammation at D10-12 and signs of clinical resolution by D28. Despite similar kinetics in the inflammatory response between young male and females, sex differences are observed in the magnitude of the transcriptional response by microglia and the adaptive component of the infiltrating response. With age, the inflammation increases and persists after AAV2 injection. Based on the microglia transcriptional response to AAV, males maintain similar signature across age, although enhanced with increasing age. Contrary, females have greater divergence in their inflammatory response across age. Of particular note, old females have enriched cellular stress and inflammatory microglia gene signatures, with corresponding retinal degeneration. These findings inform crucial sex and age differences for therapeutic application of ocular gene therapy. Our dataset highlight the need to further define these differences to appropriately tackle AAV immunogenicity for all populations.